The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner

Abstract

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application.

Fig 1. Gene organization of the two-component system and the HtrA-like proteases encoding genes.

(A) Schematic representation of the E. coli chromosomal region of the cpxRA two-component system, the gene encoding the E. coli HtrA-like protease (degP) and adjacent genes. Schematic representacion of the B.subtilis (B) and S. lividans (C) chromosomal regions of the cssRS two-component system, the HtrA-like proteases encoding genes and adjacent genes.

Fig 2. CssR binds to the regulatory regions of the genes encoding the three HtrA-like proteases.

DNA fragments containing the respective potential regulatory regions of the three HtrA-like protease genes and the cssSR operon were incubated with growing concentration of phosphorylated CssR (0, 4, 13, 26, 44 μM) and subjected to gel shift assay; the degU regulatory region was carried out as a negative control.

Fig 3. Alpha-amylase produced by S. lividans TK21(pAMI11), S. lividans ΔhtrB (pAMI11), S. lividans ΔhtrA1 (pAMI11) and S. lividans ΔhtrA2 (pAMI11).

Cell-associated and extracellular amylase present in S. lividans TK21 (pAMI11) (A), S. lividans ΔhtrB (pAMI11) (B), S. lividans ΔhtrA1 (pAMI11) (C) and S. lividans ΔhtrA2 (pAMI11) (D) were analysed at different times of growth (16, 24, 36, 48 and 60 h) by Western blot using antibodies raised against AmlB. The amount of protein loaded onto the gels was corrected by the dried weight of the bacterial cultures. Molecular size markers are indicated on the side of each panel. The arrow indicates the relative mobility of the mature AmlB.

Fig 4. Complementation of the alpha-amylase synthesised by the HtrB, HtrA1 and HtrA2 deficient strains by propagation in multicopy of their respective genes.

(A) Extracellular alpha-amylase present in the supernatants of the HtrB-deficient strain transformed with pFD666 derivative plasmids containing htrB, htrA1 and htrA2 (pFDB, pFDA1 and pFDA2, respectively) (B) HtrA1-deficient strain transformed with pFDB, pFDA1 and pFDA2 (C), HtrA2-deficient strain transformed with pFDB, pFDA1 and pFDA2 (D) and S. lividans TK21 transformed with pFDB, pFDA1 and pFDA2 were analysed at different times of growth (16, 24, 36, 48 and 60 h) by Western blotting assays using antibodies raised against AmlB. The amount of protein loaded onto the gels was corrected by the dried weight of the bacterial cultures. Molecular size markers are indicated on the side of each panel. The arrows indicate the relative mobility of the mature AmlB.

Fig 5. Predicted mode of action of the three HtrA-like proteases in S. lividans.

HtrA1 recognizes the misfolded protein outside of the cytoplasmic membrane while the complex formed by HtrB and HtrA2 remain localised in the cytoplasmic membrane. The HtrA1 PDZ domain could interact with the HtrA2 PDZ domain turning the three proteases into an active state favouring the cleavage of the misfolded extracellular overproduced proteins.